Compositions of enriched central nervous system stem cell and progenitor cell populations

ABSTRACT

Enriched neural stem and progenitor cell populations, and methods for identifying, isolating and enriching for neural stem cells using reagent that bind to cell surface markers, are provided.

CLAIM OF PRIORITY

This application is a divisional application of U.S. Ser. No.09/422,844, filed Oct. 21, 1999, now U.S. Pat. No. 6,468,794, whichclaims the benefit of U.S. Ser. No. 60/119,725, filed Feb. 12, 1999.

TECHNICAL FIELD

This invention relates generally to enriched neural stem cell andprogenitor cell populations, and methods for identifying, isolating andenriching for neural stem and progenitor cells, particularly centralnervous system neural stem cells and progenitor cells, and mostparticularly to enriched populations of neurosphere initiating cells(NS-IC).

BACKGROUND OF THE INVENTION

Stem cell populations constitute only a small percentage of the totalnumber of cells, but are of immense interest because of their ability torepopulate the body. The longevity of stem cells and the disseminationof stem cell progeny are desirable characteristics. There is significantcommercial interest in these methods because stem cells have a number ofclinical uses. There is also medical interest in the use of stem cellsas a vehicle for gene therapy.

Proteins and other cell surface markers found on stem cell andprogenitor cell populations are useful in preparing reagents for theseparation and isolation of these populations. Cell surface markers arealso useful in the further characterization of these important cells.

Yin et al., U.S. Pat. No. 5,843,633, incorporated herein by reference,describes a monoclonal antibody called AC133, which binds to a surfacemarker glycoprotein on hematopoietic stem and progenitor cells. TheAC133 antigen is a 5-transmembrane cell surface antigen with a molecularweight of 117 kDa. Expression of this antigen is highly tissue specific,and has been detected on a subset of hematopoietic progenitor cellsderived from human bone marrow, fetal bone marrow and liver, cord blood,and adult peripheral blood. The subset of cells recognized by the AC133antibody is CD34^(bright), and contains substantially all of the CFU-GMactivity present in the CD34⁺ population, making AC133 useful as areagent for isolating and characterizing human hematopoietic progenitorand stem cells.

However, surface markers specific to non-hematopoietic stem cells andprogenitor cells, and particularly central nervous system neural stemcells and progenitor cells have not been identified. Further, the AC133antibody has not been used in methods for identifying, isolating, orenriching for non-hematopoietic stem cells or progenitor cells,particularly central nervous system (CNS) neural stem cells andprogenitor cells. There remains a need for tools, such as monoclonalantibodies that are useful in isolating and characterizing humannon-hematopoietic progenitor and stem cells, and particularly centralnervous system (CNS) neural stem cells and progenitor cells.

SUMMARY OF THE INVENTION

This invention provides methods for identifying, isolating, andenriching for human non-hematopoietic progenitor and stem cells, andparticularly central nervous system (CNS) neural stem cells which caninitiate neurospheres (NS-IC) and progenitor cells. The invention alsoprovides for enriched populations containing CNS neural stem cells thatcan initiate neurospheres, and progenitor cells. A “neurosphereinitiating cell (NS-IC)” is a cell that can initiate long-termneurosphere culture. A “neurosphere”, in turn, is an aggregate orcluster of cells which includes neural stem cells and primitiveprogenitors. The identification, culture, growth, and use ofneurospheres is disclosed in Weiss et al., U.S. Pat. No. 5,750,376 andWeiss et al., U.S. Pat. No. 5,851,832, both incorporated herein byreference. While the term “NS-IC” is defined by the ability or capacityof that cell to form a neurosphere, these cells may be appropriatelygrown in adherent culture (see, for example, Johe, U.S. Pat. No.5,753,506, incorporated herein by reference), and it should be notedthat the methods and populations described herein are not to be limitedto suspension cultures of NS-IC. An NS-IC is nestin⁺ and has thecapability to differentiate, under appropriate differentiatingconditions, to neurons, astrocytes, and oligodendrocytes.

According to one embodiment of this invention, enriched populations ofnon-hematopoietic stem cells and progenitor cells, preferably CNS neuralstem cells including NS-ICs, and progenitor cells, and method ofidentifying, isolating, or enriching for such cells, is achieved bycontacting a population of cells containing at least one stem cell orNS-IC, or progenitor cell with a reagent that binds to surface markerglycoprotein antigen (“AC133 antigen”) recognized by the AC133 antibody.In a preferred embodiment the reagent is the AC133 antibody (the AC133antibody is alternately referred to herein as “5F3”). Use of traditionaltechniques for cell sorting, such as by immunoselection (e.g., FACS),then permits identification, isolation, and/or enrichment for cells inwhich contact between the reagent and the AC133 antigen has beendetected.

In another embodiment, this invention provides a novel antibody, hereincalled 5E12, that may be used to provide enriched populations ofnon-hematopoietic stem cells and progenitor cells, preferably CNS neuralstem cells that can initiate neurospheres and progenitor cells, and maybe used in methods of identifying, isolating, or enriching for suchcells, by contacting a population of cells containing at least one stemcell NS-IC, or progenitor cell with the 5E12 antibody, which binds to asurface marker glycoprotein antigen other than the AC133 antigen.

In a preferred embodiment, the cells of this invention, preferably theCNS neural stem cells, are additionally characterized as lacking cellsurface markers for CD45 and CD34.

In a further embodiment, this invention provides a novel antibody,herein called 8G1, believed to recognize CD24, which permitssubselection between populations of CNS neural stem cells (characterizedas 8G1^(−/lo)) and populations of CNS progenitor cells (characterized as8G1⁺).

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a diagram illustrating the proliferation and differentiationof a NS-IC.

FIG. 2 is a series of photographs showing that neurosphere cultures canbe initiated from single-cell sorted 5F3⁺ cells.

FIG. 3 is a dot plot of fluorescence activated cell sorting (FACS) datashowing the isolation of human CNS neural stem cells using cell surfacemarkers using the monoclonal antibody 5E12. The x axis represents cellstaining for antibodies to CD34 and CD45. The y axis represents cellstaining with the 5E12 antibody.

FIG. 4 is a two panel dot plot of FACS sorting data showing theisolation of human neural stem cells by cell surface markers. Panel Ashows that 5F3⁺ cells co-express the antigen for the 5E12 antibody.Panel B shows that 5F3⁺ cells typically do not express the antigen forthe 8G1 antibody.

FIG. 5 is a chart showing the distribution of 5F3⁺ cells in fetal brainas a function of gestational age.

FIG. 6 is a series of photographs showing results of the transplantationof human neural cells into NOD SCID mouse.

FIG. 7 is a series of photographs showing that the progeny of 5F3⁺sorted cells migrate through the rostral migratory stream (RMS) whentransplanted into a rodent model.

FIG. 8 is a series of photographs showing that the progeny of 5F3⁺sorted cells migrate through the (RMS) into the olfactory bulb whentransplanted into a rodent model.

DETAILED DESCRIPTION OF THE INVENTION

A population of cells exists within the adult central nervous system(CNS) which exhibit stem cell properties, in their ability to self-renewand to produce the differentiated mature cell phenotypes of the adultCNS. These stem cells are found throughout the CNS, and particularly inthe subventricular regions, and dentate gyms of the hippocampus.

Growth factor-responsive stem cells can be isolated from many regions ofthe neuraxis and at different stages of development, of murine, rodentand human CNS tissue. These cells vary in their response to growthfactors such as EGF, basic FGF (bFGF, FGF-2) and transforming growthfactor alpha (TGF∝), and can be maintained and expanded in culture in anundifferentiated state for long periods of time. Both adult andembryonic murine progenitor cells respond to EGF and grow as spheres ofundifferentiated cells. These cells show the characteristics of stemcells in that they are multipotent, and under serum containingconditions can differentiate into neurons, astrocytes andoligodendrocytes, as well as maintaining a subpopulation which remainsundifferentiated and continues to proliferate under EGF administration.Murine EGF-responsive progenitor cells express mRNA for the EGF receptorin vitro. Human CNS neural stem cell cultures have also been identified.The identification, culture, growth, and use of mammalian, includinghuman, neural stem cell cultures, either as suspension cultures or asadherent cultures, is disclosed in Weiss et al., U.S. Pat. No. 5,750,376and Weiss et al., U.S. Pat. No. 5,851,832, both incorporated herein byreference. Similarly, Johe, U.S. Pat. No. 5,753,506, incorporated hereinby reference, refers to adherent CNS neural stem cell cultures. Whencultured in suspension, CNS neural stem cell cultures typically formneurospheres.

FIG. 1 is shows the proliferation of a NS-IC as it develops into aneurosphere, and subsequent differentiation into neuronal and glialphenotypes, as well as generation of a progeny NS-IC. In the presence ofone or more proliferation-inducing growth factors, the NS-IC divides andgives rise to a sphere of undifferentiated cells composed of more stemcells and progenitor cells (a “neurosphere”). When the clonally derivedneurosphere is dissociated and plated as single cells, in the presenceof one or more proliferation-inducing growth factors, each NS-IC cangenerate a new neurosphere. The cells of a single neurosphere are clonalin nature because they are the progeny of a single neural stem cell. Inthe continued presence of a proliferation-inducing growth factor such asEGF or the like, precursor cells within the neurosphere continue todivide resulting in an increase in the size of the neurosphere and thenumber of undifferentiated neural cells. The neurosphere is notimmunoreactive for glial fibrillary acidic protein (GFAP; a marker forastrocytes), neurofilament (NF; a marker for neurons), neuron-specificenolase (NSE; a marker for neurons) or myelin basic protein (MBP; amarker for oligodendrocytes). However, cells within the neurosphere areimmunoreactive for nestin, an intermediate filament protein found inmany types of undifferentiated CNS cells (Lehndahl et al., 60 CELL585–595 (1990), incorporated herein by reference). Antibodies areavailable to identify nestin, including the rat antibody referred to asRat401. If the neurospheres are cultured in conditions that allowdifferentiation, the progenitor cells differentiate to neurons,astrocytes and oligodendrocytes. The mature phenotypes associated withthe differentiated cell types that may be derived from the neural stemcell progeny are predominantly negative for the nestin phenotype.

The terminology used for undifferentiated, multipotent, self-renewing,neural cells has evolved such that these cells are now termed “neuralstem cells.” A neural stem cell is a clonogenic multipotent stem cellwhich is able to divide and, under appropriate conditions, hasself-renewal capability for NS-IC and can include in its progenydaughter cells which can terminally differentiate into neurons,astrocytes, and oligodendrocytes. Hence, the neural stem cell is“multipotent” because stem cell progeny have multiple differentiationpathways. A neural stem cell is capable of self maintenance, meaningthat with each cell division, one daughter cell will also be on averagea stem cell.

The non-stem cell progeny of a neural stem cell are typically referredto as “progenitor” cells, which are capable of giving rise to variouscell types within one or more lineages. The term “neural progenitorcell” refers to an undifferentiated cell derived from a neural stemcell, and is not itself a stem cell. Some progenitor cells can produceprogeny that are capable of differentiating into more than one celltype. For example, an O-2A cell is a glial progenitor cell that givesrise to oligodendrocytes and type II astrocytes, and thus could betermed a “bipotential” progenitor cell. A distinguishing feature of aprogenitor cell is that, unlike a stem cell, it does not exhibit selfmaintenance, and typically is thought to be committed to a particularpath of differentiation and will, under appropriate conditions,eventually differentiate into glia or neurons.

The term “precursor cells” refers to the progeny of neural stem cells,and thus includes both progenitor cells and daughter neural stem cells.

Cell markers. This invention provides for the identification, isolation,enrichment, and culture of neural stem cells that are capable of formingneurospheres (NS-IC). NS-ICs are identified or selected through thebinding of antigens, found on the surfaces of NS-ICs, to reagents thatspecifically bind the cell surface antigen.

One of these antigens is an antigen that binds to the AC133 monoclonalantibody. The AC133 antibody (herein referred to as the 5F3 antibody) isexemplary of antibody embodiments of reagents that recognize a humancell marker termed prominin. Prominin is a polytopic membrane proteinexpressed in various epithelial cells (Weigmann et al., 94(23) Proc NatlAcad Sci USA. 12425–30 (1997); Corbeil et al., 112 (Pt 7) J Cell Sci.1023–33 (1999); Corbeil et al., 91(7) Blood 2625–6 (1998); Miriglia etal., 91(11) Blood 4390–1 (1998)). Various AC133 antibodies are describedin U.S. Pat. No. 5,843,333, incorporated herein by reference. A depositof the murine hybridoma cell line AC133 was made at the American TypeTissue Collection, 12301 Parklawn Drive, Rockville Md. 20852, on Apr.24, 1997, and given the ATCC designation HB12346. These AC133 antibodiesare capable of immunoselection for the subset of human cells of interestin this invention. Preferred AC133 monoclonal antibodies can be obtainedcommercially from Miltenyi Biotec Inc. (Auburn Calif.), includingAC133/1-PE antibody (Cat #808-01) and AC133/2-PE antibody (Cat #809-01).For MACS separation, a 50:50 mixture of the monoclonal antibodies ispreferred. The high tissue specificity of AC133 expression isparticularly advantageous during enrichment for highly purified NS-ICpopulations.

5E12 is a novel monoclonal antibody generated againstenzymatically-dissociated human fetal brain cells. The 5E12 monoclonalantibody was generated substantially according to the contralateralimmunization method described in Yin, U.S. Pat. No. 5,843,633,incorporated herein by reference. The antigen to which 5E12 binds has aputative MW 125 kD, and is currently believed to be a distinct antigenfrom prominin.

CD45 is the T200/leucocyte common antigen. Antibodies to CD45 arecommercially available. In a preferred embodiment, the cells of thisinvention and cultures containing them, are additionally characterized(in addition to being prominin positive) as lacking cell surface markerssuch as CD45.

CD34 is also known as gp105-120. Monoclonal antibodies to CD34 arecommercially available, and CD34 monoclonal antibodies have been used toquantitate and purify lymphohematopoietic stem/progenitor cells forresearch and for clinical bone marrow transplantation.

The monoclonal antibody 8G1 is believed to recognize CD24 (antibodies toCD24 are commercially available), and specifically reacts with the 515kilodalton α-chain of human LRP/A2MR which is expressed in a restrictedspectrum of cell types. A strong immunohistochemical reaction is seen inhepatocytes, tissue macrophages, subsets of neurons and astrocytes inthe central nervous system, fibroblasts, smooth muscle cells, andmonocyte-derived foam cells in atherosclerotic lesions in the arterialwall. The antibody can also be used for the characterization of a subsetof myelomonocytic subtypes of chronic and acute leukemia (CD91).Antibodies to CD91 are commercially available.

Cell Deposit. The 8G1.7 and 5E12.5 subject cultures have been depositedwith the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209, on Dec. 20, 1999, under ATCC accessionnumbers PTA-993 and PTA-994, respectively, in accordance with theprovisions of the Budapest Treaty for the Deposit of Microorganisms.Accordingly, upon granting of a patent, all restrictions on theavailability of the deposited materials will be irrevocably removed.

Isolation, enrichment, and selection of cells. The population of cellsfrom which NS-ICs are isolated can be a neural tissue, a population ofcells is dissociated from neural tissue, or a population of cells incell culture, e.g., a cells in a neurosphere culture or an adherentneural stem cell culture.

The invention provides for the isolation and identification of NS-ICs.Identification of a neurosphere initiating stem cell (NS-IC) involvescontacting a population of neural cells (or which contains neural orneural derived cells) with a reagent that binds to AC133 antigen anddetecting the contact between the reagent that binds to AC133 antigenand the AC133 antigen on the surface of cells. Those cells to which thereagent binds are identified as NS-ICs. The identity of those cells canbe confirmed by assays to demonstrate that the cells are in fact NS-ICs,capable of neurosphere initiation, self renewal and multipotentcy.

The methods of this invention can also be used to isolate AC133⁺ cellsfrom AC133⁻ cells using an AC133 antibody, by combining a population ofneural cells which contains a fraction of NS-ICs with a reagent thatspecifically binds to the AC133 antigen, and then selecting for AC133⁺cells, to produce a selected population enriched in AC133⁺ NS-ICs ascompared with the population of neural cells before selection.

Accordingly, the invention further provides for the enrichment of NS-ICsfrom neural tissue or neural stem cell cultures (e.g., neurospheresuspension cultures or neural stem cell adherent cultures). Theinvention is thus useful for the enrichment of NS-IC from neural tissuein which stem cells and progenitor cells occur at low frequency, or mayhave been depleted, such as late embryo, juvenile, and adult tissue. Oneof skill in the art can combine a population of neural cells containinga fraction of NS-ICs with a reagent that specifically binds to the AC133antigen; and select for the AC133⁺ cells. In this way, the selectedAC133⁺ cells are enriched in the fraction of NS-IC as compared with thepopulation of neural cells.

The cell selection can be by any suitable means known in the art,including flow cytometry, such as by fluorescence activated cell sortingusing a fluorochrome conjugated AC133 antibody. The selection can alsobe by high gradient magnetic selection using AC133 antibody isconjugated to magnetic particles. Any other suitable method includingattachment to and disattachment from solid phase, is also contemplatedwithin the scope of the invention.

One of skill in the art can derive the population of cells byimmunoselection using an AC133 antibody. The population of cells shouldcontain at least 30% AC133⁺ NS-ICs, preferably at least 50–70% AC133⁺NS-ICs, and more preferably greater than 90% AC133⁺ NS-ICs. Mostpreferable would be a substantially pure population of AC133⁺ NS-ICs,comprising at least 95% AC133⁺ NS-ICs. The degree of enrichmentobtained, and actually used, depends on a number of factors, includingthe method of selection, the method of growth, and the cell dose of thecells that are placed in culture for the initiation of neurospheres.

The population of cells can be derived from late embryo, juvenile, oradult mammalian central nervous system (CNS) tissue, or it may bederived from existing cultures of neural stem cells, as described inWeiss, U.S. Pat. No. 5,750,376, or Johe, U.S. Pat. No. 5,753,506. In themost preferred embodiment, the NS-IC are human. In some embodiments, theAC133⁺ cells in the population can be complexed to endothelial cells.

The in vitro cell cultures described herein containing an enrichedpopulation of AC133⁺ NSICs are generally characterized in that thecultures stain positive for nestin and, in the presence ofdifferentiation-inducing conditions, produce progeny cells thatdifferentiate into neurons, astrocytes, and oligodendrocytes.

One of skill in the art can introduce an isolated AC133⁺ cell to aculture medium, proliferate the isolated AC133⁺ cell in culture;particularly as a neurosphere; culture the progeny of the isolatedAC133⁺ cell under conditions in which the isolated AC133⁺ celldifferentiates to neurons, astrocytes, and oligodendrocytes; then detectthe presence of neurons, astrocytes, and oligodendrocytes. The presenceof neurons, astrocytes, and oligodendrocytes characterizes the isolatedAC133⁺ cell as an NS-IC.

Typically AC133⁺ NS-IC is cultured in a medium that permits the growthand proliferation of neurospheres. The culture in which the isolatedAC133⁺ cell proliferates can be a serum-free medium containing one ormore predetermined growth factors effective for inducing multipotentneural stem cell proliferation. The culture medium can be supplementedwith a growth factor selected from leukocyte inhibitory factor (LIF),epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2;bFGF) or combinations thereof. The culture medium can be furthersupplemented with neural survival factor (NSF) (Clonetics, Calif.). Theconditions in which the AC133⁺ cell differentiates to neurons,astrocytes, and oligodendrocytes can be culturing the AC133⁺ cellprogeny on a laminin-coated surface in culture medium containing fetalbovine serum (FBS) without EGF, FGF-2 or LIF.

The invention also provides a method for identifying the presence of agrowth factor that affects the growth of NS-IC. One of skill in the artcombines a composition suspected of containing at least one growthfactor that affects the growth of NS-IC with a composition comprisingNS-IC, then determines the growth of the NS-IC as a function of thepresence of the composition. Altered (increased, decreased, etc.) NS-ICgrowth indicates the presence in the composition of a growth factor thataffects the growth of NS-IC. One can then further identify the growthfactor.

Antibodies to AC133. Antibodies to AC133 may be obtained or prepared asdiscussed in U.S. Pat. No. 5,843,633, incorporated herein by reference.The AC133 antigen can be contacted with an antibody, such as variousAC133 monoclonal antibodies, which have specificity for the AC133antigen. An AC133 antibody is characterized by binding to the AC133protein under Western blot conditions from reducing SDS-PAGE gels. TheAC133 antigen has a molecular weight, based on commercially availablestandards, in the range of about 117 kDa. The AC133 antigen is expressedon a subset of progenitor cells derived from human bone marrow, fetalbone marrow and liver, cord blood, and adult peripheral blood.

Antibodies to AC133 antigen can be obtained by immunizing a xenogeneicimmunocompetent mammalian host (including murine, rodentia, lagomorpha,ovine, porcine, bovine, etc.) with human progenitor cells. The choice ofa particular host is primarily one of convenience. A suitable progenitorcell population for immunization can be obtained by isolating CD34⁺cells from cytokine mobilized peripheral blood, bone marrow, fetalliver, etc. A suitable progenitor cell population for immunization canbe obtained from CNS neural stem cells or other NS-IC. Immunizations areperformed in accordance with conventional techniques, where the cellsmay be injected subcutaneously, intramuscularly, intraperitoneally,intravascularly, etc. Normally, from about 10⁶ to 10⁸ cells will beused, which may be divided up into 1 or more injections, usually notmore than about 8 injections, over a period of from about one to threeweeks. The injections may be with or without adjuvant, e.g. complete orincomplete Freund's adjuvant, specol, alum, etc.

After completion of the immunization schedule, the antiserum may beharvested in accordance with conventional ways to provide polygonalantisera specific for the surface membrane proteins of progenitor cells,including the AC133 antigen. Lymphocytes are harvested from theappropriate lymphoid tisue, e.g. spleen, draining lymph node, etc., andfused with an appropriate fusion partner, usually a myeloma line,producing a hybridoma secreting a specific monoclonal antibody.Screening clones of hybridomas for the antigenic specificity of interestis performed in accordance with conventional methods.

AC133 antibodies can be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al., 269 J. BIOL. CHEM. 26267–73 (1994), incorporated herein byreference, and others. DNA sequences encoding the variable region of theheavy chain and the variable region of the light chain are ligated to aspacer encoding at least about 4 amino acids of small neutral aminoacids, including glycine or serine. The protein encoded by this fusionallows assembly of a functional variable region that retains thespecificity and affinity of the original antibody.

AC133 antibodies can be produced by use of Ig cDNA for construction ofchimeric immunoglobulin genes (Liu et al., 84 PROC. NATL. ACAD. SCI.3439 (1987) and 139 J. IMMUNOL. 3521 (1987), incorporated herein byreference. mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al., “Sequences of Proteins of Immunological Interest” N.I.H.PUBLICATION NO. 91-3242 (1991). Human C region genes are readilyavailable from known clones. The chimeric, humanized antibody is thenexpressed by conventional methods.

AC133 antibodies can be produced as antibody fragments, such as Fv,F(ab′)₂ and Fab. Antibody fragments may be prepared by cleavage of theintact protein, e.g. by protease or chemical cleavage. Alternatively, atruncated gene is designed. For example, a chimeric gene encoding aportion of the F(ab′)₂ fragment would include DNA sequences encoding theCH1 domain and hinge region of the H chain, followed by a translationalstop codon to yield the truncated molecule.

Immunostaining. Biological samples are assayed for the presence ofAC133⁺ cells by any convenient immunoassay method for the presence ofcells expressing the surface molecule bound by the subject antibodies.Assays may be performed on cell lysates, intact cells, frozen sections,etc. The antibodies available from Miltenyi Biotec Inc. (Auburn Calif.)are suitable for the direct immunofluorescent staining of cells.

Cell sorting. The use of cell surface antigens to NS-IC cells provides ameans for the positive immunoselection of progenitor cell populations,as well as for the phenotypic analysis of progenitor cell populationsusing flow cytometry. Cells selected for expression of AC133 antigen maybe further purified by selection for other stem cell and progenitor cellmarkers.

For the preparation of substantially pure progenitor and stem cells, asubset of progenitor cells is separated from other cells on the basis ofAC133 binding. Progenitor and stem cells may be further separated bybinding to other surface markers known in the art.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography and “panning”with antibody attached to a solid matrix, e.g. plate, or otherconvenient technique. Techniques providing accurate separation includefluorescence activated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. Dead cellsmay be eliminated by selection with dyes associated with dead cells(propidium iodide [PI], LDS). Any technique may be employed which is notunduly detrimental to the viability of the selected cells.

Conveniently, the antibodies are conjugated with labels to allow forease of separation of the particular cell type, e.g. magnetic beads;biotin, which binds with high affinity to avidin or streptavidin;fluorochromes, which can be used with a fluorescence activated cellsorter; haptens; and the like. Multi-color analyses may be employed withthe FACS or in a combination of immunomagnetic separation and flowcytometry. Multi-color analysis is of interest for the separation ofcells based on multiple surface antigens, e.g. AC133⁺ CD45⁻, AC133⁻CD34⁺, etc. Fluorochromes which find use in a multi-color analysisinclude phycobiliproteins, e.g. phycoerythrin and allophycocyanins;fluorescein and Texas red. A negative designation indicates that thelevel of staining is at or below the brightness of an isotype matchednegative control. A dim designation indicates that the level of stainingmay be near the level of a negative stain, but may also be brighter thanan isotype matched control.

In one embodiment, the AC133 antibody is directly or indirectlyconjugated to a magnetic reagent, such as a superparamagneticmicroparticle (microparticle). Direct conjugation to a magnetic particleis achieved by use of various chemical linking groups, as known in theart. Antibody can be coupled to the microparticles through side chainamino or sufhydryl groups and heterofunctional cross-linking reagents. Alarge number of heterofunctional compounds are available for linking toentities. A preferred linking group is 3-(2-pyridyidithio)propionic acidN-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle.

Alternatively, AC133 antibody is indirectly coupled to the magneticparticles. The antibody is directly conjugated to a hapten, andhapten-specific, second stage antibodies are conjugated to theparticles. Suitable haptens include digoxin, digoxigenin, FITC,dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugationof the hapten to a protein, i.e. are known in the art, and kits for suchconjugations are commercially available.

To practice the method, the AC133 antibody is added to a cell sample.The amount of AC133 Ab necessary to bind a particular cell subset isempirically determined by performing a test separation and analysis. Thecells and AC133 antibody are incubated for a period of time sufficientfor complexes to form, usually at least about 5 min, more usually atleast about 10 min, and usually not more than one hr, more usually notmore than about 30 min.

The cells may additionally be incubated with antibodies or bindingmolecules specific for cell surface markers known to be present orabsent on progenitor or stem cells.

The labeled cells are separated in accordance with the specific antibodypreparation. Fluorochrome labeled antibodies are useful for FACSseparation, magnetic particles for immunomagnetic selection,particularly high gradient magnetic selection (HGMS), etc. Exemplarymagnetic separation devices are described in WO 90/07380,PCT/US96/00953, and EP 438,520. The AC133 Cell Isolation Kit (MiltenyiBiotec Inc., Auburn Calif.) can be used for the positive selection ofAC133⁺ cells. The kit provides a tool for single step isolation ofAC133⁺ cells. The AC133 Cell Isolation Kit contains FcR Blocking Reagentand MACS colloidal MicroBeads conjugated to the monoclonal mouseanti-human AC133 antibody.

The purified cell population may be collected in any appropriate medium.Various media are commercially available and may be used, includingDulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution(HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove'smodified Dulbecco's medium (IMDM), phosphate buffered saline (PBS) with5 mM EDTA, etc., frequently supplemented with fetal calf serum (FCS),bovine serum albumin (BSA), human serum albumin (HSA), etc.

Populations highly enriched for human progenitor or stem cells areachieved in this manner. The desired cells will be 30% or more of thecell composition, preferably 50% or more of the cell population, morepreferably 90% or more of the cell population, and most preferably 95%or more (substantially pure) of the cell population.

Use of purified stem cell/progenitor cells. The AC133⁺ stemcells/progenitor cells are useful in a variety of ways. The AC133⁺ cellscan be used to reconstitute a host whose cells have been lost throughdisease or injury. Genetic diseases associated with cells may be treatedby genetic modification of autologous or allogeneic stem cells tocorrect a genetic defect or treat to protect against disease.Alternatively, normal allogeneic progenitor cells may be transplanted.Diseases other than those associated with cells may also be treated,where the disease is related to the lack of a particular secretedproduct such as hormone, enzyme, growth factor, or the like. CNSdisorders encompass numerous afflictions such as neurodegenerativediseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g.stroke, head injury, cerebral palsy) and a large number of CNSdysfunctions (e.g. depression, epilepsy, and schizophrenia). In recentyears neurodegenerative disease has become an important concern due tothe expanding elderly population which is at greatest risk for thesedisorders. These diseases, which include Alzheimer's Disease, MultipleSclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, andParkinson's Disease, have been linked to the degeneration of neuralcells in particular locations of the CNS, leading to the inability ofthese cells or the brain region to carry out their intended function. Byproviding for maturation, proliferation and differentiation into one ormore selected lineages through specific different growth factors theprogenitor cells may be used as a source of committed cells.Neurospheres can also be used to produce a variety of blood cell types,including myeloid and lymphoid cells, as well as early hematopoieticcells (see, Bjornson et al., 283 SCIENCE 534 (1999), incorporated hereinby reference).

The AC133⁺ cells may also be used in the isolation and evaluation offactors associated with the differentiation and maturation of cells.Thus, the cells may be used in assays to determine the activity ofmedia, such as conditioned media, evaluate fluids for growth factoractivity, involvement with dedication of lineages, or the like.

The AC133⁺ cells may be frozen at liquid nitrogen temperatures andstored for long periods of time, being thawed and capable of beingreused. The cells will usually be stored in 5% DMSO and 95% fetal calfserum. Once thawed, the cells may be expanded by use of growth factorsor stromal cells associated with stem cell proliferation anddifferentiation.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. These examples should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLE 1 AC133 Magnetic Cell Sorting (MACS) Positive Selected FetalBrain Cells Contain Neurosphere Initiating Cell (NS-IC) Activity

AC133⁺ cells are prepared by the following method: Human fetal brain(FBR 10–20 gestational week [“g.w.”]) were obtained from AdvancedBioscience Resources, INC (Oakland, Calif.) after obtaining informedconsent. Human fetal brain tissues were cut into 1–3 mm cubed piecesusing scalpels, transferred into 50 mL centrifuge tubes and wash oncewith 0.02% EDTA/PBS solution. Tissues were dissociated enzymatically inthe presence of collagenase and hyaluronidase at 37° C. for 1 hr. Debrisand aggregates were removed by filtering cell suspensions through 70micron filter cup.

AC133⁺ human fetal brain cells were separated by using paramagneticantibody microbeads, AC133/1 Cell isolation Kit (Cat. # 508-01, MiltenyiBiotec, Auburn, Calif.). MACS separations were performed based oninstruction accompanied with the kit. In a representative flowcytometric MACS separation from a typical AC133⁺ isolation, about 44% ofthe cells were AC133⁺ CD45⁻, while about 2% were CD34⁺ (These CD34⁺cells were endothelial cells complexed to the purified NS-IC).

The AC133⁺ selected cells which resulted from the method described above(using brain 18 g.w.) were still heterogeneous. The cells tended to forma complex with endothelial cells. Endothelial cells were identified asCD34⁺ or CD105⁺. AC133 MACS separation also enriches CD34⁺ endothelialcells which are associated with AC133⁺ cells. (After passaging, theNS-IC separate from the complexed endothelial cells and purified NS-ICcan be obtained.)

AC133⁺ MACS separated cells were cultured in the presence of mediacontaining EGF, FGF-2, and LIF, as described above. In general, cellsfrom early gestational age fetal brain (5–12 g.w.) were enriched forNS-IC and no enrichment was required to initiate neurosphere cultures.On the other hand, cells from older fetal brain samples (16–20 g.w.)contained far less NS-IC activity and required enrichment for initiatingneurosphere cultures. In other words, AC133⁺ is useful for theenrichment of NS-IC from older gestational age humans brain tissue.AC133⁺ MACS separated cells from fetal brain (18 g.w.) were enriched forNS-IC activity, while whole human fetal brain cells (18 g.w.) withoutAC133⁺ MACS separation failed to initiate neurosphere cultures.

Neurosphere cells established from AC133 MACS cells express nestin, astested after ˜7 days in culture and detected by rabbit anti-human nestinpolyconal antibodies. For example, among the neurosphere cells availablefrom CytoTherapeutics (Providence, R.I.), FBR 1069 (18 g.w.) and FBR1070 (20 g.w.) expressed nestin. When induced to differentiate, theAC133⁺ MACS-derived neurosphere cells could differentiate into neuronsand astrocytes, as detected by β-tubulin staining for neurons and GFAPstaining for astrocytes. In this particular differentiation assay,neurosphere cells were cultured onto a laminin-coated surface in thepresence of 1% FBS and without EGF, FGF-2 and LIF.

Other differentiation assay can be used to induce differentiation ofNS-IC to neurons, astrocytes and oligodendrocytes.

EXAMPLE 2 AC133 is a Critical Cell Surface Marker Expressed on Cellsfrom Long-Term Neurosphere Culture

A long-term neurosphere cell culture, 8.5 FBR, was obtained fromCytoTherapeutics Inc. (Providence, R.I.). The 8.5 FBR neurosphere cellsexpress AC133 relatively uniformly. These 8.5 FBR cells are also Thy-1⁺,CD166⁺, and HLA-DR⁺. When Ex Vivo 15 was used as basal media, higherpercentage of neurosphere cultures initiated from primary brain tissuefrom 18 g.w. It is therefore possible to evaluate AC133⁺ fraction ofcells in developing neurosphere cultures. The proportion of AC133⁺ cellsincreased as neurosphere developed. Once neurosphere cells were wellestablished, virtually all cells forming neurospheres expressed AC133.

EXAMPLE 3 Neurosphere Initiating Cells Can be Separated Using MonoclonalAntibody, AC133: Flow Cytometry Cell Sorting (FACS) Approach

The purpose of this EXAMPLE is to test whether AC133⁺ cells were theonly cells in the brain that have pluripotent NSC activity. mAb againsthuman CD45 was used to exclude blood cell contamination in fetal tissue.In some cases, mAb against human CD34 was also used to excludeendothelial cells and endothelial-neural progenitor complexes. The fetalbrain cells were thus defined as CD45⁻ CD34⁻. To measure neural stemcells and primitive progenitor activities, a NS-IC assay was establishedto determine frequency of NS-IC in a given population. When NS-IC arerare and express AC133 antigen uniformly, NS-IC can be enriched byAC133⁺ selection, and correspondingly depleted in other fractions.

Source of monoclonal antibodies: AC133 antigen was defined by two mAbsAC133/1 and AC133/2, both conjugated with phycoerythrin, which areavailable through Miltenyi Biotec (Auburn, Calif.). Anti human CD45-FITCand Glycophrin A-FITC were obtained from CALTAG (Burlingame, Calif.) andCoulter (Miami, Fla.) respectively. Anti-humanallophycocyanin-conjugated CD34 was obtained from BDIS (San Jose,Calif.).

Cell preparation: FBR were dissociated by collagnease and hyaluronidase,and still contained endothelial-progenitor complex, which prevented theisolation of a candidate of NSC in single cell suspension (endothelialcells are CD45⁺). To dissociate this endothelial cell-NS-IC complex, FBRcells processed as described above, were further treated with trypsinfor 10–15 min. The AC133 antigen, CD45 antigen and CD34 antigen wereresistant for trypsin treatment, while Glycophrin A was sensitive.

After trypsin digestion, cells were washed and stained with mAbs againstCD45, Glycophrin A, AC133 and CD34. No immunomagnetic bead selectionswere used. The cells were incubated for 20–60 min on ice. After thefinal wash, the cells were resuspended in HBSS solution containing 1μg/mL propidium idodine (PI). The labeled cells were analyzed and sortedwith a dual-laser FACS (Becton Dickinson, San Jose). Dead cells wereexcluded from analysis by their PI staining characteristics. Aftersorting purity of sorted cell populations were checked by FACSreanalysis. A representative FACS profiles of before sorting andpost-sorting of AC133⁺ CD45⁻ cells (NS-IC, ˜5% of the starting cells)and AC133⁻ CD45⁻ cells (˜87% of the starting cells) was performed.

NS-IC activity is highly enriched in the AC133⁺ but not the AC133⁻subset: FBR cells (typically 16–20 g.w.) were typically sorted for CD45⁻CD34⁻ AC133⁻ and CD45⁻ CD34⁻ AC133⁺ fractions. No significant NS-ICactivity resided in CD45⁺ or CD45⁻CD34⁺ populations in FBR.

The sorted cells were cultured in the neurosphere media described above.Typically, Ex Vivo 15 or combination of Ex Vivo 15, D-MEM, F-12 mediawere used as a basal medial. To maximize neurosphere development, thesorted cells were typically cultured in the presence of LIF, FGF-2, EGFand neural survival factor, NSF (Cat. CC-4323, Clonetics, San Diego,Calif.).

A single cell suspension was obtained after cell sorting. After 4–5 daysin vitro culture, the AC133⁺ cells started to proliferate and smallneurospheres were observed 8–10 days post culture initiations. The cellscould initiate neurospheres when cultured in the presence of LIF, FGF-2,EGF without NSF. Neurosphere cultures were initiated from four out offour FBR tissues (18–20 g.w.) sorted for AC133⁺ CD45⁻ or AC133⁺ CD45⁻CD34⁻.

In contrast, when AC133⁻ CD45⁻ FBR cells were placed in culture in thepresence of LIF, FGF-2, and EGF, very few neurosphere formations wereseen, and failed to passage to a new flask. When additional NSF wasadded in the growth media, some neurosphere initiation was observed.Thus, AC133⁻ CD45⁻ FBR cells were depleted in a significant amount ofNS-IC.

EXAMPLE 4 AC133⁺ Cell Separation to Enrich for NS-IC Cells

AC133⁺ cell separation can effectively be used to enrich for NS-IC cellsfrom tissue. Furthermore AC133⁺ cell separation can further enrich forNS-IC cells from established preparations. In one test, AC133⁺ cellsorting of dissociated neurospheres (CytoTherapeutics, Providence, R.I.)provides a greatly enriched NS-IC culture and shows increasedneurosphere establishment. Using that culture, the cell dose required toinitiate a neurosphere in each well (i.e., 100% positive) can be reducedfrom 3,000–10,000 cells to about 30 cells (see, TABLE 1, below).

TABLE 1 Cell % % Tissue ID Cell Dose Score #Well Positive Negative FBR1104 Post trypsin 1,000 6 24 25.0% 75.0% Ex Vivo 15 3,000 20 24 83.3%16.7% LIF/EGF/ 10,000 24 24 100.0% 0.0% FGF-2 30,000 12 12 100.0% 0.0%100,000 12 12 100.0% 0.0% FBR 1104 Post trypsin 1,000 23 24 95.8% 4.2%Ex/Vivo 15 3,000 24 24 100.0% 0.0% LJF/EGF/ 10,000 24 24 100.0% 0.0%FGF-2/NSF 30,000 12 12 100.0% 0.0% 100,000 12 12 100.0% 0.0% FBR 1104AC133 neg. 1,000 0 24 0.0% 100.0% selected cells Ex Vivo 15 3,000 1 244.2% 95.8% LIF/EGF/ 10,000 28 48 58.3% 41.7% FGF-2/NSF AC133⁺ 10 2 248.3% 91.7% selected cells Ex Vivo 15 100 24 24 100.0% 0.0% LIF/EGF/ 30024 24 100.0% 0.0% FGF-2/NSF 1,000 24 24 100.0% 0.0% FBR 1101 Posttrypsin 1,000 9 24 37.5% 62.5% Ex Vivo 15 3,000 16 24 66.7% 33.3%LIF/EGF/ 10,000 23 24 95.8% 4.2% FGF-2 30,000 12 12 100.0% 0.0% 100,00012 12 100.0% 0.0% FBR 1101 Post trypsin 1,000 16 24 66.7% 33.3% Ex Vivo15 3,000 21 24 87.5% 12.5% LIF/EGF/ 10,000 24 24 100.0% 0.0% FGF-2/NSF30,000 12 12 100.0% 0.0% 100,000 12 12 100.0% 0.0% FBR 1101 AC133⁺ 1 996 9.4% 90.6% selected cells Ex Vivo 15 10 42 60 70.0% 30.0% LIF/EGF/ 3023 24 95.8% 4.2% FGF-2/NSF 100 11 12 91.7% 8.3%

As shown in TABLE 1, the non-enriched fresh brain tissue (“FBR”) usedhere (g.w. 20) may contain NS-IC in such numbers that it requires a celldose of between 3,000 and 10,000 cells to initiate neurospheres in everywell. By using the method of the invention, enriched populations can beobtained, such that a cell dose of 1,000 cells or less is required, andmore preferably an enriched population such that a cell dose of lessthan 100 cells is required. As shown in TABLE 1, enrichment here hasbeen achieved so that a cell dose of only about 30 cells is required perwell to establish a neurosphere culture in each well. TABLE 1 also showsthat when populations are depleted in AC133⁺ cells (FBR 1104 AC133 neg.selected cells), establishment of neurosphere cultures from thosepopulations is markedly reduced.

Quantitative NS-IC assay: To assay for the presence of NS-IC, cellpopulations suspected of containing the multipotent NS-IC are subjectedto clonal development. Cells are grown in proliferation medium to formneurospheres, then induced to differentiate to form neurons, astrocytes,and oligodendrocytes. The presence of neurons, astrocytes, andoligodendrocytes can be shown by immunostaining. For example, neuronsstain for the presence of β-tubulin; astrocytes stain for the presenceof GFAP; and oligodendrocytes stain for the presence of O4.

The quantitative NS-IC assay can be performed on unpurified tissuecells, on AC133⁺ sorted cells, and on clonal neurosphere cell lines.

EXAMPLE 5 Cell Culture Media For Growth and Passage of NS-IC

Weiss et al., U.S. Pat. No. 5,750,376 and Weiss et al., U.S. Pat. No.5,851,832 disclose “culture medium containing one or more predeterminedgrowth factors effective for inducing multipotent neural stem cellproliferation” and “differentiation-inducing conditions” However,different basal media can be used, including, but not limited to:

D-MEM/F12 (Gibco BRL, Gaithersburg, Md.);

Ex Vivo 15 (Bio Whittaker, Walkersville, Md.);

Neural progenitor basal media, (Clonetics. San Diego, Calif.); or

combination of the basal media listed above.

A typical media formulation to culture human neurosphere cells isprovided in TABLE 2.

TABLE 2 Serum-Free N2/EGF Supplemented Culture Medium For NeurospheresQuantity Reagents 87 ml DMEM/F12 (Gibco lot. 1012915; Cat. No.11330-032) 1 ml N-2 Supplement (Gibco lot 1017018; Cat. No. 17502-014) 1ml 0.2 mg/ml heparin (Sigma lot 28H0320; Cat. No. H-3149) 1 ml 0.2MGlutamine (JCR lot 7N2320; Cat. No. 59202-77p) 10 ml 3% Glucose (Sigma,lot 37H0841; Cat. No. G-7021) 20 μl 100 μg/ml EGF (R&D lot CE107091;Cat. No. 236-EG) 100 μl 20 μg/ml FGF-2 (Gibco lot KCQ411; Cat. No.13256-029) 100 μl 10 μg/ml LIF (R&D lot OX038021; Cat. No. 250-L)

EGF is added to 100 ml base medium for human neurospheres afterfiltering the medium. EGF is relatively stable in the medium. FGF-2 andLIF are added when medium is ready to use. The final concentrations ofthe supplement reagents are:

5 μg/ml Insulin 100 μg/ml Human transferrin 6.3 ng/ml Progesterone 16.1μg/ml Putrascine 5.2 ng/ml Selenite 20 ng/ml EGF 20 ng/ml FGF-2 10 ng/mlLIF 2 μg/ml heparin 2 mM L-glumtamine 6 mg/ml Glucose

The optimization of media formulation permits a higher percentage ofneurospheres initiated from primary brain tissue to be established. Weprefer Ex Vivo 15 media. The optimization of media formulation alsopermits a more consistent growth of neurospheres. To maximizeneurosphere development, the NS-IC are typically cultured in thepresence of LIF, bFGF, EGF and neural survival factor, NSF (Cat.CC-4323, Clonetics, San Diego, Calif.). In one test, both trypsinizedFBR 1101 neural cells and trypsinized FBR 1104 neural cells(CytoTherapeutics, Providence, R.I.), show increased growth whencultured in Ex Vivo 15 medium with LIF, bFGF, EGF, and NSF.

EXAMPLE 6 Direct Isolation of Human Neural Stem Cells From Fetal Brainby Cell Sorting

A large source of highly defined engraftable human cells capable ofextensive neuronal regeneration could be an effective therapeuticproduct for the treatment of neurodegenerative disorders. To definereproducible methods for the enrichment of human neural stem cells(NSCs), we have developed and used monoclonal antibodies (mAbs) directedtoward surface markers on human neural cells to identify and purify NSCsby fluorescence activated cell sorting (FACS). Based on FACS andimmunohistochemical analyses, two mAbs, 5F3 and 5E12 were identified.They defined small subsets of fetal brain cells and displayed specificreactivity to cells in the floor plate and ependymal layer of the spinalcord (12 g.w), sites known to contain CNS stem cells. These mAbs, stainless than 5% of FBR cells, and greater than 95% of cells from long-termneurosphere cultures were positive.

As an example, two cell populations 5F3+CD34−CD45−(5F3+) and5F3−CD34−CD45−(5F3−) were sorted and tested for their ability toinitiate neurosphere cultures. The 5F3+ subset was highly enriched forneurosphere-initiating cell activity; they proliferated to form smallneurosphere by 8–10 days in culture. In contrast, the sorted 5F3− cellsremained as a single cell suspension, failed to initiate neurospheres,and eventually died. The expanded 5F3+ sorted neurosphere cells werepositive for nestin expression, and differentiated into neurons and gliafollowing exposure to differentiation conditions. Using the NOD SCIDmouse, in vivo studies show that at 8 weeks post transplantation the5F3+ neurosphere cells can engraft and migrate. These studies show thatwe have identified and enriched human NSCs based on cell surface markersand flow cytometry and demonstrated their activity using in vitro andvivo assays.

In further experiments, we examined brain and spinal cord tissues overvarious gestational ages. The earlier (5–12 wk gestation) gestationalages have a higher frequency of neurosphere initiating cell (NS-IC) thanlater gestational ages (16–20 wk gestation). See, e.g., FIG. 5. Directculturing of cells derived from these tissues leads to neurosphereinitiation.

Our data (shown in the Table below) demonstrate that cell population ofneural cells enriched for 5F3⁺ cells are enriched for NS-IC activity, asmuch as 23 fold.

Population % in brain NS-IC Range Post processed 100 1/819 1/304–1435brain cells (n = 8) 5F3 − sorted (n = 6) 95 1/5434 1/4224–7772 5F3 +sorted (n = 6) 4.6 1/36 1/10–74

Further, as FIG. 2 shows, neurospheres can be derived from single-cellsorted 5F3⁺ cells. We have also demonstrated that self-renewal ofneurosphere cells derived from 5F3⁺ sorted cells can be achieved byre-initiation of neurospheres from single cells (data not shown).Conversely, our data indicates that cell populations depleted of 5F3⁺cells are also depleted for NS-IC activity.

EXAMPLE 7 Isolation of NS-IC By Different Markers

As a second example, we sorted cell populations using a novel monoclonalantibody, 5E12, described herein. The 5E12⁺ subset was enriched forneurosphere-initiating cell activity, as shown in the Table below. Seealso FIG. 3. Our data suggests that the antigen to the 5E12 antibody iscoexpressed with the AC133 antigen on 5F3⁺ cells.

We also evaluated the 8G1 monoclonal antibody as a subselector forneural stem cells, as shown in the Table below. Cells that were 5F3⁺ and8G1^(−/lo) displayed more stem cell-like properties, while cells thatwere 5F3⁺ and 8G1^(med/hi) displayed more progenitor cell-likeproperties.

ENRICHMENT OF NS-IC BY 5F3, 5E12 AND 8G1 ANTIBODIES Population % inbrain NS-IC Range Brain cells 100 1/819 1/304–1435 control (n = 8) 5F3 −sorted (n = 6) 95 1/5434 1/4224–7772 5F3 + (n = 6) 4.6 1/36 1/10–74 5E12− (n = 2) 97 1/1335 1/1259,1411 5E12 + (n = 3) 2.5 1/286 1/79–392 5F3 +8G1^(−/lo) (n = 3) 1.1 1/23 1/15–34 5F3 + 8G1^(mid/hi) (n = 3) 1.7 1/631/38–105 *All sorted populations were CD34⁻ CD45⁻

EXAMPLE 9 In Vivo Studies NS-IC

We transplanted 5F3+ sorted NS-ICs (obtained as described above) intothe lateral ventricles of neonatal immunodeficient mice, usingconventional techniques. Engraftment and migration of human neurospherecells were detected between 4–8 weeks after injection using a humanspecific Thy-1 antibody (see FIG. 6). As shown in FIG. 7, staining withhuman β-tubulin (a neuronal marker) and human nuclear antigen (forlocalization of human cells) revealed migration of the human neurospherecells through the rostral migratory stream (RMS). Further, as shown inFIG. 8, localization using human nuclear antigen demonstrated that humanneurosphere cells had migrated through the RMS to the olfactory bulb.

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the invention to the preciseform disclosed, but by the claims appended hereto.

1. A composition comprising: (a) a population of neural cells enrichedfor neurosphere initiating stem cells (NS-IC) produced by: (1) combininga population comprising neural cells or neural-derived cells containinga fraction of NS-ICs with monoclonal antibody AC133 or monoclonalantibody 5E12 (ATCC Accession No. PTA-994); and (2) selecting andisolating those cells that bind to monoclonal antibody AC133 ormonoclonal antibody 5E12, thereby producing a population of cellsenriched for NS-IC as compared with the population of neural cells orneural-derived cells; and (b) at least one monoclonal antibody selectedfrom the group consisting of monoclonal antibody AC133 and monoclonalantibody 5E12, wherein the at least one monoclonal antibody isconjugated with a label to allow for ease of separation.
 2. An in vitrocell culture composition comprising: (a) a population of neural cellsenriched in NS-IC, wherein the NS-IC bind to monoclonal antibody AC133or to monoclonal antibody 5E12 (ATCC Accession No. PTA-994) and areCD45⁻ cells; (b) a medium capable of supporting the growth of the cells;and (c) at least one monoclonal antibody selected from the groupconsisting of monoclonal antibody AC133 and monoclonal antibody 5E12 ,wherein the at least one monoclonal antibody is conjugated with a labelto allow for ease of separation.
 3. An in vitro cell culture compositioncomprising: (a) a population of neural cells enriched in NS-IC, whereinthe NS-IC bind to monoclonal antibody AC133 or to monoclonal antibody5E12 (ATCC Accession No. PTA-994) and are CD45⁻ CD34⁻ cells; (b) amedium capable of supporting the growth of the cells; and (c) at leastone monoclonal antibody selected from the group consisting of monoclonalantibody AC133 and monoclonal antibody 5E12, wherein the at least onemonoclonal antibody is conjugated with a label to allow for ease ofseparation.
 4. An in vitro cell culture composition comprising: (a) apopulation of neural cells enriched in NS-IC, wherein the NS-IC bind tomonoclonal antibody AC133 or to monoclonal antibody 5E12 (ATCC AccessionNo. PTA-994) and are CD24⁻ cells; (b) a medium capable of supporting thegrowth of the cells; and (c) at least one monoclonal antibody selectedfrom the group consisting of monoclonal antibody AC133 and monoclonalantibody 5E12, wherein the at least one monoclonal antibody isconjugated with a label to allow for ease of separation.
 5. Thecomposition of any one of claims 1 and 2–4, further comprising a solidsupport to which the cells are attached.
 6. The composition of any oneof claims 1 and 2–4, wherein the population of cells comprises at least70% cells that bind to monoclonal antibody AC133 or monoclonal antibody5E12.
 7. The composition of any one of claims 1 and 2–4, wherein thepopulation of cells comprises at least 90% cells that bind to monoclonalantibody AC133 or monoclonal antibody 5E12.
 8. The composition of anyone of claims 1 and 2–4, wherein the population of cells that bind tomonoclonal antibody AC133 or monoclonal antibody 5E12 is a substantiallypure population.
 9. The composition of any one of claims 1 and 2–4,wherein the medium comprises a serum-free medium containing one or moregrowth factors effective for inducing multipotent neural stem cellproliferation.
 10. The composition of any one of claims 1 and 2–4,wherein the medium comprises a growth factor selected from the groupconsisting of leukocyte inhibitory factor (LIF), epidermal growth factor(EGF), basic fibroblast growth factor (FGF-2), and combinations thereof.11. The composition of any one of claims 1 and 2–4, wherein the mediumcomprises neural survival factor, NSF.
 12. The composition of any one ofclaims 1 and 2–4, wherein the neural cells are human.
 13. Thecomposition of any one of claims 1 and 2–4, wherein the label isselected from the group consisting of magnetic beads, magnetic reagents,superparamagnetic microparticles, biotin, fluorochromes, and haptens.